Human platelets are activated at sites of vascular injury by the combined effects of multiple agonists, most of which activate platelets via G protein coupled receptors. The G proteins that are normally expressed in platelets include three ubiquitous members of the G1 family (Gi1, Gi2 and Gi3) and one (Gz), that is expressed most prominently in platelets and brain. Our previous goals for this project included identifying unique roles for Gz in platelet activation, identifying receptors and effectors that interact with Gz, identifying sites of phosphorylation in the a subunit of Gz, and analyzing the promoter region of the gene encoding Gzalpha. Substantial progress was made towards all of these goals, culminating with the development of Gzalpha knockout mice and the demonstration that Gz is required for the potentiation of platelet aggregation and the suppression of cAMP formation by physiological concentrations of epinephrine. In addition, we have recently observed that Gz and other Gi family members in platelets are needed for maximal activation of the low molecular weight GTP-binding protein, Rap1B, and obtained data implicating Rap1B in platelet aggregation. Based on these results, the studies in this proposal will continue our efforts to understand the role of all four G family members in platelet activation. It is our hypothesis that the biochemical properties that distinguish the various Gi family members determine how they are regulated, which receptors and effectors they interact with, and, ultimately, the efficiency with which different agonists cause platelet activation. The proposed studies are divided into four specific aims. The first will address the differences in receptor and effector coupling among the Gi family members and the role of Gi-derived Gbetagamma. The second aim will examine the regulatory impact of the phosphorylation of Gzalpha by protein kinase C and the Rac1/cdc42-activated kinase, PAK, during platelet activation. The third aim will extend our studies on the regulation of Rap1B in platelets by Gi family members and the fourth will examine the regulation of cAMP formation in circulating platelets. All four of these aims will combine biochemical and pharmacologic approaches with studies on genetically engineered mice lacking selected receptors and G proteins, allowing us to better understand platelet activation in vivo.